Analysis of RNA polymerase-promoter complex formation.
Bacterial promoter identification and characterization is not as straightforward as one might presume. Promoters vary widely in their similarity to the consensus recognition element sequences, in their activities, and in their utilization of transcription factors, and multiple approaches often must be used to provide a framework for understanding promoter regulation. Characterization of RNA polymerase-promoter complex formation in the absence of additional regulatory factors (basal promoter function) can provide a basis for understanding the steps in transcription initiation that are ultimately targeted by nutritional or environmental factors. Promoters can be localized using genetic approaches in vivo, but the detailed properties of the RNAP-promoter complex are studied most productively in vitro. We first describe approaches for identification of bacterial promoters and transcription start sites in vivo, including promoter-reporter fusions and primer-extension. We then describe a number of methods for characterization of RNAP-promoter complexes in vitro, including in vitro transcription, gel mobility shift assays, footprinting, and filter binding. Utilization of these methods can result in determination of not only basal promoter strength but also the rates of transcription initiation complex formation and decay.
Ross
Wilma
W
Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Dr., Madison, WI 53706, USA. weross@wisc.edu
Gourse
Richard L
RL
D001419:Bacteria / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D024363:Transcription Initiation Site; D015533:Transcriptional Activation / Q000235:genetics
D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases